ANTIBACTERIAL EFFECTS OF SINGLE AND COMBINED CRUDE EXTRACTS OF SYNADENIUM GLAUCESCENS AND COMMIPHORA SWYNNERTONII

Background: Synadenium glaucescens and Commiphora swynnertonii are among the reported plants used traditionally for treatment of bacterial infections. This study reports antibacterial effects of single and combined extracts from leaves, stem and root barks of Commiphora swynnertonii and Synadenium glaucescens. Materials and Methods: Plants were collected from Manyara and Njombe regions in Tanzania. Extraction was done using dichloromethane and methanol. The extracts were assessed for antibacterial activity against Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Klebsiella pneumonia and Pseudomonas aeruginosa). Minimum Inhibitory Concentrations (MIC) was determined by broth microdilution, while Fractional Inhibitory Concentration (FIC) indices were calculated from MIC values of combined extracts to determine combination effects. Results: Strong antibacterial activities were demonstrated by all extracts of S. glaucescens (MIC 0.011-0.375mg/mL) against Gram-positive bacteria and methanol extracts of C. swynnertonii (MIC 0.047-0.375mg/mL). Synergistic effect was observed when combining methanol extracts of C. swynnertonii stem bark with S. glaucescens leaves against S. aureus (∑FIC 0.5), Other synergistic effects were observed against E. faecalis with dichloromethane extracts of C. swynnertonii stem bark and S. glaucescens stem bark (∑FIC 0.5), and C. swynnertonii root bark and S. glaucescens root bark (FIC index 0.3). For the remaining combinations, mainly additive effects were observed. Conclusion: Synergistic effects on bacteria were observed by combining different plant parts of S. glaucescens and C. swynnertonii suggesting that it could be beneficial to combine such extracts when used for antibacterial purposes.


Introduction
Herbal products have been used as medicines since the commencement of human life (Masimba et al., 2014). The recipes for medicinal plant preparation for the treatment of several ailments are evidenced from the earliest Sumerian, Indian, Egyptian, and Chinese publications (Karunamoorthi et al., 2013). Unlike pharmaceuticals, where the ingredients are well defined and characterized, herbal products contain multiple bioactive compounds with little or no understanding of how these compounds function, likewise the effect of herbal combinations is usually poorly characterized (Gupta et al., 2017). When herbal combinations are administered together there is a possibility of causing chemical or pharmacological effects that may increase or decrease the effectiveness or severity of adverse effects via synergistic, additive, or antagonistic effects (Shi and Klotz, 2012;Sheng et al., 2018). In Tanzania, people access a variety of medicines to meet their healthcare needs. At least 70% of the population is estimated to use traditional medicines (Stanifer et al., 2015). Synadenium glaucescens (Mvunjakongwa in Swahili) and a tropical tree Commiphora Swynnertonii (Oltemwai in Maasai) which belong to the families Euphorbiaceae and Burseraceae respectively are among the medicinal plants used by Tanzanians to treat various diseases in humans (Bakari et al., 2012;Mabiki et al., 2013;Mkangara et al., 2014). These plants contain secondary metabolites such as alkaloids, flavonoids, phenols, terpenoids, anthraquinones, steroids, and essential oils (Mabiki et al., 2013;Kalala et al., 2014). Such compounds are reported to have activity against infections caused by bacteria, fungi, viruses, and pests in humans and livestock (Bakari et al., 2012;Mabiki et al., 2013;Mkangara et al., 2014). Despite the exhibited potentials of some individual herbal drugs in the treatment of some infectious diseases, there are reported failures of most single drugs or medicines in the treatment of many pathogenic infectious diseases (Wang et al., 2021). The root causes of these hindrances are reported to be the development of anti-microbial resistance, a narrow antimicrobial spectrum, and limited activity of antimicrobials agents (Rubaka et al., 2014;Ayukekbong et al., 2017). As a result, these failures may cause an increase in the number of morbidities, mortality, disability, and socioeconomic costs (Stanifer et al., 2015). Therefore, there is a need for the search for novel antibacterial drugs from natural resources like herbs to combat the reported hindrances for antimicrobial activities (Bhardwaj et al., 2016). Due to synergistic effects resulting between the combination of more than one drugs in the treatment of microbial infections, it has been reported to be the best techniques to fight against hindrances for antimicrobial effects (Vuuren and Viljoen, 2011). Hence, this study focused on evaluation of antibacterial activities of combined extracts from leaves, stem barks, and root barks of S. glaucescens and C. swynnertonii. The results from this study, especially for the combinations which demonstrated synergistic effects, may be adopted for the treatment of bacterial infections. However, further study on safety for these combinations is highly recommended.

Study design and study Area
This study was an experimental one where the antibacterial effects of combinations of herbal medicines were assessed based on their effects and efficacies against selected bacteria. The study was conducted in the chemistry laboratory, Department of Chemistry and Physics, and microbiology laboratory, Department of Biosciences, of the College of Natural and Applied Sciences of the Sokoine University of Agriculture (SUA).

Plant collection and preparation
The leaves, stem, and root barks of Synadenium glaucescens were collected from Mtulingala village in Njombe region coordinates 08 o 34' to 08 o 49' S and 08 o 34' to 03 o 55' E meters above sea level. The root barks, leaves, and stem barks of Commiphora swynnertonii were collected from Mirerani-Simanjiro District in Manyara region coordinates 03 o 36' to 03 o 14.73' S and 36 o 50' to 36 o 18.05' E meters above the sea level. Plant parts were washed with clean water then peeled to separate the barks and wood. Plant materials were dried in a dark room at 20 o C at the Tanzania Tree Seed Agency Laboratory, Morogoro. Dry samples were grounded separately using a lab mill machine (Christy Hunt Engineering Ltd, England) to obtain approximately 2mm particle size. The selection of these plant parts was based on the previously conducted studies on antimicrobial activity against selected bacteria (Max et al., 2014;Mkangara et al., 2014).

Reagents
Solvents used for extraction and dissolving sample in this study were methanol (Finer Chemical, Gujarat-India), dichloromethane, and dimethyl Sulphoxide (Loba Chemie, Mumbai-India). The standard antibiotic used as positive control was gentamicin (Sigma-Aldrich, Germany).

Extraction and Concentration
Extraction of extracts were carried out using the method used by Bakari et al. (2012) and Max et al. (2014). Briefly, 1000g of dry ground plant materials were extracted by dichloromethane using hot continuous extraction method at 50 o C for 4 hours whereby 33g of dry ground samples were injected into each thimble (33mm diameter, 80mm length) and extracted using Soxhlet apparatus. The samples were filtered and the obtained solid residues were soaked in methanol at room temperature (25-30 o C) for 72 hours. All samples were filtered using Whatman No.1 filter paper (Maidstone-Kent, UK). The filtrates were concentrated in a rotary evaporator (Buchi Labortetechnik, Flawil, Switzerland) with a bath maintained at 40 o C. The obtained crude extracts were air-dried to remove remains of solvents. The Dried extracts were stored in a refrigerator at 6 o C until further use.

Test bacterial strain
Gram-positive bacteria used were Staphylococcus aureus American Type Culture Collection (ATCC 29213) and Enterococcus faecalis (ATCC 51559). Gram-negative bacteria used were Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 1145), Pseudomonas aeruginosa (ATCC 27853). These belong to species that are major causes of nosocomial infections, and where antimicrobial resistance is a high treat to human health (WHO, 2002).

Preparation of individual and combined crude extracts solutions
A stock concentration of 3 mg/ml crude extract from leaves, stem barks and root barks of S. glaucescens and C. Swynnertonii was made. Depending on the MIC value of each crude extract, the different concentrations were made to make working bench solutions. The extracts were combined in ratio 1:1v/v, 1: 1:1v/v and 1:1:1:1v/v.

Minimum inhibitory concentrations (MIC) by broth dilution method
MIC values were determined by a two-fold microdilution method to assess the antibacterial effects of herbherb combinations according to Kudumela et al. (2018). In brief, sterile, 96-well polystyrene microtiter plates was first preloaded with 50µL of Mueller Hinton broth in each well followed by the addition of 50µL of extract solutions into the first well of each row to make a total volume of 100µL. Each of the test sample materials was tested in duplicate. To the first well, the samples were mixed and 50µL was drawn from each well and transferred to the subsequent wells until the last wells. Then 50µL of the mixture from the last well was discarded. Thereafter, 50µL of the bacterial suspension equivalent to 0.5 MacFarland standard turbidity (1.5×106 CFU mL-1) was added to each well. An additional row containing 0.1mg/ml of gentamicin (50µL) was used as a positive control. Wells containing (50µL) solvent and bacteria only were used as negative controls. The plates were incubated at 37°C overnight. MIC was determined visually, whereby the lowest concentration without growth of bacteria was considered as the MIC.

Fractional inhibitory concentration (FIC)
Checkerboard assay was employed to determine the Fraction Inhibitory Concentration (FIC) as described (Jain et al., 2011). FIC is determined by a methodology similar to that utilized for the determination of MIC, however modified so that it is useful to test the antibacterial activities of combinations of extracts (Meletiadis et al., 2010). The summation of fractional inhibitory concentration (ΣFIC) was calculated for each tested sample independently as specified in the following algebraic formula (Kudumela et al., 2018).
Where the combined effect, was interpreted as synergistic if the FIC index ≤0.5, additive if 0.5 > FIC Index < 4, or antagonistic if FIC Index ≥ 4. This interpretation follows the conventional model suggested by (Odds, 2003) and Kassim et al., (2016). 888

Antibacterial activity of individual extracts
The evaluations of antibacterial activities of individual extracts were conducted and the MIC of each extract was obtained as indicated in Table 1 and 2. The MIC values were interpreted based on classification criteria as follows; 0.05-0.5mg/mL strong activity, 0.6-1.5mg/mL moderate activity and above 1.5mg/mL weak activity . Among the crude extracts tested, methanol extracts of leaves, stem barks and root barks of S. glaucescens and C. swynnertonii inhibited the growth of gram-positive bacteria S. aureus and E. faecalis considerable with the lowest MIC values range 0.011 -0.375mg/mL as shown in Table 1 and 2. Dichloromethane extracts of S. glaucescens and C. swynnertonii showed moderate antibacterial activity against Gram-positive bacteria tested with MIC values range 0.75mg/mL-1.5mg/mL. Furthermore, all extracts showed weak activity against Gram-negative bacteria (Tables1 and 2). However, gentamicin showed stronger antibacterial activity than the extracts tested (Tables1 and 2).

Antibacterial activity of combined crude extracts and fractional inhibitory concentrations
The combination effects were evaluated with respect to MIC value of each crude extract against bacteria. In the combination of 1:1v/v, the extracts exhibited strong activity against Gram-positive bacteria S. aureus and E. faecalis with MIC values ≤0.5 (Table 3) However, crude extracts combined in ratios 1:1:1 and 1:1:1:1v/v revealed moderate activity against S. aureus with MIC values range 0.6-1.5mg/mL (Table 3). Additionally, these combinations exhibited weak antimicrobial activity with MIC values above 1.5 mg/mL (Table 3) against the tested gram-negative bacteria E. coli, K. pneumoniae and P. aeruginosa. The FIC values were calculated and antibacterial effects were outlined in Table 4. In 1:1v/v combinations, One (1) synergistic effect observed in combination of methanol extracts of C. swynnertonii stem barks and S. glaucescens leaves against S. aureus (∑FIC 0.5) ( Table 4). Other two synergistic effects were observed against E. faecalis in dichloromethane extracts of C. swynnertonii stem barks and S. glaucescens stem barks (∑FIC 0.5), and C. Swynnertonii root barks and S. glaucescens root barks with FIC index 0.3 (Table 4). Furthermore, three (3) antagonistic effects were observed in the combinations of dichloromethane leaves extract of C. swynnertonii and root barks of S. glaucescens, stem barks of C. swynnertonii and root barks of S. glaucescens, and root barks of C. swynnertonii and root barks of S. glaucescens against. S. aureus with FIC Index values 6, 19, and 38 (Table 4). In addition, other antagonistic effects were observed against E. faecalis in combinations of methanol leaves extract of C. swynnertonii and S. glaucescens leaves, stem barks of C. swynnertonii, and root barks of S. glaucescens, and leaves of C. swynnertonii and root barks of S. glaucescens with FIC Index values 10 and 19 ( Table 4). The 1:1:1v/v and 1:1:1:1v/v combination ratios revealed antagonistic effects against S. aureus and additive effects against E. faecalis (Table 4). Moreover, the extracts in the combination ratio of 1:1v/v and 1:1:1v/v tested against Gram-negative bacteria revealed additive effects with FIC Index value 2 (Table 4), whereby the extracts in the combination ratio of 1:1:1:1:1v/v showed different antagonistic effects against Gram-negative bacteria with FIC Index values 4, 5, 6 and 8 ( Table 4).

Antibacterial activity of individual and combined crude extracts
Herbal medicines are normally prepared either singly or in combination with several plant species (Vuuren and Viljoen, 2011). In this study, crude extracts from leaves, stem barks, and root barks of C. swynnertonii and S. glaucescens were screened for antibacterial properties both individually and in combinations against selected bacteria. The findings of this study for the individual plant parts of C. swynnertonii are in agreement with previous studies reported by Bakari  Furthermore, a previous study conducted by Max et al. (2014) for the crude root extract of S. glaucescens reported antibacterial activity against S. aureus and moderate activity against P. aeruginosa. Similarly, in the current study individual methanol extracts of the parts of S. glaucescens showed strong activity against S. aureus and E. faecalis.
In this study, however, the individual extracts of these plant parts displayed weak activity against Gramnegative bacteria tested. The difference in susceptibility for Gram-positive bacteria and Gram-negative-bacteria may be associated with differences in their cell wall structure. Gram-negative bacteria are reported to be more resistant due to impermeability/efflux of their outer membrane/cell wall which acts as a barrier to many environmental substances including herbal drugs or antibiotics (Rawat and Nair, 2010).
Moreover, this study reports the antibacterial effects of combined crude extracts of S. glaucescens and C. swynnertonii. It is clear from Table 4 that there is a greater antibacterial activity in some combined extracts than individual extracts. The combined extracts which showed synergistic effects may be promising alternatives for antibacterial therapy in the future, and their effects should be investigated further. However, in this study additive effects were also demonstrated in several combinations (Table 4). This effect occurs when the activity of the combined extracts is equivalent to the sum of the activity of each extract when used individually (Adams et al., 2006). This effect signifies that the biological actions of the combined extracts interact with similar molecular targets or metabolic pathways (Vuuren and Viljoen, 2011). Antagonistic effects were also observed in some combinations against the tested bacteria (Table 4). This indicates that, the extracts have conflicting effect that may block or reduce the effectiveness of one or both extracts. Usually, this type of effect is discouraged for therapeutic application (Bassolé and Juliani, 2012).

Conclusion
Combined extracts of S. glaucescens and C. swynnertonii have additive effects against gram-positive bacteria tested. Further, combined extracts of root barks of C. swynnertonii and stem barks of S. glaucescens have synergistic effect against gram-positive bacteria tested, suggesting that it can be advantageous to combine such extracts to form their products.
Therefore, based on the combinations which showed synergistic effects against some of the tested bacteria, this study provides promising alternative herbal antimicrobials from plants. However, it is recommended that further studies on the combinations that showed synergistic effects should be carried out on their toxicity and mode of action to optimize their use.